Polyscias filicifolia (Araliaceae) Hairy Roots with Antigenotoxic and Anti-Photogenotoxic Activity

Hairy root cultures are considered as a valuable source of bioactive phytoconstituents with expanding applicability for their production. In the present study, hairy root cultures of Polyscias filicifolia (Araliaceae), a traditional Southeast Asian medicinal plant, were established. The transformation with Agrobacterium rhizogenes ATCC 15834 allowed to obtain 15 root lines. The K-1 line, demonstrating the highest growth capabilities, was subjected to further investigations. To enhance the biosynthetic potential of hairy roots, methyl jasmonate elicitation approach was applied (MeJA; at different doses and exposure time), with subsequent transfer of elicited roots to control medium. This strategy resulted in chlorogenic acid production up to 1.59 mg/g dry weight. HPLC-PDA-ESI-MS analysis demonstrated variation in extracts composition and allowed to identify different caffeic and ferulic acid derivatives. Next, cytotoxic, antigenotoxic, and anti-photogenotoxic properties of hairy roots extracts were determined. None of the tested extracts were cytotoxic. In addition, they demonstrated significant antigenotoxic activity with the highest protective potential; up to 52% and 49% of inhibition of induction ratio (IR) induced by the 2-aminoanthracene was revealed for extracts derived from hairy roots elicited for 3 days with 50 µM MeJA and roots elicited for 7 days with 100 µM MeJA and then transferred for 30 days to control medium, respectively. These same extracts exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of chloropromazine-induced genotoxicity.


Introduction
Plant biotechnological methods offer the possibility for production of a wide array of various bioactive compounds for pharmaceutical, as well as food and cosmetic industries in a manner independent of environmental conditions, at the same time ensuring protection of biodiversity of natural resources [1]. Among various systems of cultivated in vitro cell and organs, hairy root cultures are recognized as a reasonable system for efficient production of complex specific metabolites, as they are capable of producing the same profile of metabolites as a native plant, or even de novo synthesizing biomolecules [2][3][4][5]. Many methods are used in plant biotechnology to increase the efficiency of the production of specific metabolites in cells, tissues, and organs of plants grown in in vitro cultures. One of these methods is elicitation. In response to elicitor stress, the production of plant-specific metabolites increases. Specific metabolites resulting from elicitation are mainly responsible    The addition of acetosyringone (Acs) to the MS medium had a beneficial effect on the transformation efficiency. In the case of shoots incubated on MS medium with Acs, the percentage of explants on which roots were formed was the highest and amounted to 65%. These roots emerged directly (without the callus phase) 4 weeks after the inoculation. The average number of roots developed per shoot explant was 4.8. In comparison, the transformation efficiency of explants cultivated on the MS medium without the addition of Acs was found to be 20%, with the average number of emerging roots per shoot explant being 2.9 ( Table 1).
The transformation efficiency of plantlets derived from somatic embryos was lower than that of shoots. On the MS medium supplemented with Acs, root formation was found at the site of infection in 25% of infected plants; the average number of emerging roots per explant was 2.5. On the MS medium without the addition of Acs, only 5% of the plants demonstrated root formation, and the average number of roots per explant was 1.7 (Table 1).
Finally, 15 root lines were obtained, and each root that sprouted was cut off and cultivated separately as a single line. Among all obtained root lines, only three were selected for further research: K-1, K-3, and K-5 lines ( Figure 2). Observation of the growth and morphology of these three root lines through three passages led to the final selection of the one K-1 line that was characterized by an abundant hairy zone without any callus formation. The roots of the other 12 lines did not sustain growth due to necrosis. In addition, the formation of an abundant callus was observed on the roots of the K-3 and K-5 lines. The transformation process was confirmed by PCR analysis ( Figure S1, Supplementary Materials).

Optimalisation of Culture Conditions
The most hairy root growth-promoting medium was selected on the basis of root growth index, their anatomical traits, and color. Among the tested medium modifications, the DCR I medium with L-glutamine 500 mg/L and 30 g/L sucrose proved to be the most growth promoting (Figures 3 and 4). During cultivation, the medium hairy roots of K-1 line demonstrated a growth index up to 480.8% ± 23.63. Roots were white, had numerous side branches, and formed a dense clump. These roots did not change color throughout the duration of the passage ( Figure 3).

Optimalisation of Culture Conditions
The most hairy root growth-promoting medium was selected on the basis of root growth index, their anatomical traits, and color. Among the tested medium modifications, the DCR I medium with L-glutamine 500 mg/L and 30 g/L sucrose proved to be the most growth promoting (Figures 3 and 4). During cultivation, the medium hairy roots of K-1 line demonstrated a growth index up to 480.8% ± 23.63. Roots were white, had numerous side branches, and formed a dense clump. These roots did not change color throughout the duration of the passage (Figure 3).
After various modifications of WPM medium, the root growth was significantly lower compared to the DCR I medium. The modification of WPM medium by the addition of L-glutamine 500 mg /L resulted in an improvement of root growth capacities in comparison to the WPM medium without this amino acid ( Figure 3). Contrary to the DCR I medium, with all the modifications of the WPM media, regardless of the presence of Lglutamine and at high sucrose concentration (40 g/L), the root darkening started after 3 weeks of culture and they became dark brown after 6 weeks.     [%] is calculated as follows: (final weight-initial weight/initial weight) × 100%. The same letter indicates a statistically significant difference (p < 0.05).

Phytochemical Analysis
Among four qualitatively investigated phenolic acids by the HPLC-UV-Vis analysis, only the presence of chlorogenic acid (CGA) in extracts prepared from hairy roots cultivated in control conditions, as well as in elicited cultures, was revealed ( Figure 5).
In control untreated cultures, the CGA biosynthesis was significantly lower than in elicited cultures. In addition, the increase in CGA accumulation was correlated with the time of culture ( Figure 5). The significantly (p < 0.05) highest content of CGA (1.59 ± 0.06 mg/g DW) was determined and elicited for 1 day with methyl jasmonate (MeJA) 100 µM culture followed by subsequent cultivation in control DCR I medium. It was over 12% higher concentration than that detected in hairy roots treated for 7 days with MeJA 100 µM and then cultivated for 30 days in control DCR I medium. The content of CGA in hairy roots elicited with MeJA 50 or 100 µM for 3 or 7 days, respectively, was also higher than in control unelicited culture, although no statistically significant differences in CGA concentration were observed. is calculated as follows: (final weight-initial weight/initial weight) × 100%. The same letter indicates a statistically significant difference (p < 0.05).
After various modifications of WPM medium, the root growth was significantly lower compared to the DCR I medium. The modification of WPM medium by the addition of L-glutamine 500 mg /L resulted in an improvement of root growth capacities in comparison to the WPM medium without this amino acid ( Figure 3). Contrary to the DCR I medium, with all the modifications of the WPM media, regardless of the presence of L-glutamine and at high sucrose concentration (40 g/L), the root darkening started after 3 weeks of culture and they became dark brown after 6 weeks.

Phytochemical Analysis
Among four qualitatively investigated phenolic acids by the HPLC-UV-Vis analysis, only the presence of chlorogenic acid (CGA) in extracts prepared from hairy roots cultivated in control conditions, as well as in elicited cultures, was revealed ( Figure 5).
In control untreated cultures, the CGA biosynthesis was significantly lower than in elicited cultures. In addition, the increase in CGA accumulation was correlated with the time of culture ( Figure 5). The significantly (p < 0.05) highest content of CGA (1.59 ± 0.06 mg/g DW) was determined and elicited for 1 day with methyl jasmonate (MeJA) 100 µM culture followed by subsequent cultivation in control DCR I medium. It was over 12% higher concentration than that detected in hairy roots treated for 7 days with MeJA 100 µM and then cultivated for 30 days in control DCR I medium. The content of CGA in hairy roots elicited with MeJA 50 or 100 µM for 3 or 7 days, respectively, was also higher than in control unelicited culture, although no statistically significant differences in CGA concentration were observed.
The HPLC-PDA-ESI-MS analysis of hairy root methanol extracts derived from unelicited and elicited cultures revealed variation in the qualitative profile of the samples ( Figure 6; Table 2). The major constituents of investigated extracts were caffeic and ferulic acid derivatives (Table 3). For all standard samples, mass spectra and fragmentation mass spectra were acquired for identification and confirmation of compounds presented in the methanolic extracts. In that case, a HRMS experiment was also used for confirmation of molecular formula. For the unknown compounds a HRMS experiment was the only one method, which was applied for prediction of the most likely molecular formula. In all HRMS experiments, a difference between theoretical and measurement m/z value was below 5 ppm.  The HPLC-PDA-ESI-MS analysis of hairy root methanol extracts derived from unelicited and elicited cultures revealed variation in the qualitative profile of the samples ( Figure 6; Table 2). The major constituents of investigated extracts were caffeic and ferulic acid derivatives (Table 3). For all standard samples, mass spectra and fragmentation mass spectra were acquired for identification and confirmation of compounds presented in the methanolic extracts. In that case, a HRMS experiment was also used for confirmation of molecular formula. For the unknown compounds a HRMS experiment was the only one method, which was applied for prediction of the most likely molecular formula. In all HRMS experiments, a difference between theoretical and measurement m/z value was below 5 ppm.

Cytotoxicity of Hairy Root Extracts
None of tested samples decreased BALB/c 3T3 cells' viability after 24 h treatment under 70% (Figures 7 and 8). Moreover, none of tested samples induced changes in the cells morphology in comparison to the untreated control. Therefore, all of the tested samples were classified as non-cytotoxic in vitro in the whole range of tested concentrations. Due to the lack of cytotoxicity, the IC50 values that represent the concentrations that decreased the number of living cells to 50% in comparison to the untreated control, were not calculated. However, there were observed slight but statistically significant (Student's t-test, p < 0.05) differences in the cells' viability compared with the negative control for the highest concentrations of several samples (the bars with asterisks on the Figure 7). This effect was not observed for any solutions prepared from samples collected after 50 days of the in vitro plant culture. Furthermore, on the basis of obtained results, there were no clearly visible differences among samples prepared with various solvents (methanol, 50% methanol or deionized water). The results obtained for the solvent control confirmed the lack of its influence on the cells viability. Moreover, the results obtained for the positive control fulfilled the required conditions (IC50 = 115 µg/mL) [21].
The results obtain for the presented samples and their dilution series (concentrations from 50 to 0.78 µg/mL; results unshown) did not differ from the viability of cells observed in the untreated control (Student's t-test, p < 0.05).

Antigenotoxic and Anti-Photogenotoxic Activities
The antigenotoxic and anti-photogenotoxic potentials of methanolic extracts prepared from hairy roots cultivated under various conditions were investigated. The dry residue of extracts were dissolved in methanol, 50% methanol, and deionized water. Three types of solvents were used to test whether they would affect the activity due to different degrees of dissolution of the compounds. Furthermore, methanol is toxic to the bacteria used in the umu test. The use of water as a solvent allowed higher concentrations of extracts to be tested.
In the preliminary umu-test, the photogenotoxicity and genotoxicity of the tested extracts was evaluated. None of the tested concentrations exhibited a genotoxic effect (IR < 1.5). The results of the umu-test performed with UV radiation against CPZ-induced photogenotoxicity are presented in Table 4 and Figure 9. Root extracts were added to the mutagen at concentrations of 100-800 µg/mL (extracts dissolved in water) or 12.5-100 µg/mL (dissolved in methanol or 50% methanol). Lower concentrations of the solutions were prepared in the case where the solvent was methanol, because of the toxicity of methanol against bacteria. In the highest tested concentration of water solutions ( Table 4), all of the extracts were active against CPZ. Extracts F and H exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of CPZ-induced genotoxicity. In the lowest tested concentrations of water solutions (100 µg/mL), all of the extracts were active as well, and the most active was extract I. When extracts were dissolved in methanol (Table 4), in all tested concentration extracts, G, H, and I exhibited anti-photogenotoxic potential. Extract F was not active only in the concentration of 25 µg/mL. In the case of extract C, the increase of genotoxicity was observed in the concentration of 50 µg/mL. The other tested extracts have no significant activity in the methanolic solutions. When extracts were dissolved in 50% methanol (Table 4), only four of them exhibited activity (F, G, H and I), and extract F had the strongest protective effect against CPZ-induced photogenotoxicity. A comparison of all the extracts tested at the same concentration (100 µg/mL) using different solvents is shown in Figure 9.

Antigenotoxic and Anti-Photogenotoxic Activities
The antigenotoxic and anti-photogenotoxic potentials of methanolic extr prepared from hairy roots cultivated under various conditions were investigated. The residue of extracts were dissolved in methanol, 50% methanol, and deionized water. Th

Antigenotoxic and Anti-Photogenotoxic Activities
The antigenotoxic and anti-photogenotoxic potentials of methanolic ext prepared from hairy roots cultivated under various conditions were investigated. The residue of extracts were dissolved in methanol, 50% methanol, and deionized water. T   Each value is expressed as mean ± standard deviation from at least three independent biological evaluations. Bold values denote statistical significance at the p < 0.05 level. A dash "-" denotes that the concentration has not been applied. The extracts from the following treatments were investigated: extract C, the increase of genotoxicity was observed in the concentration of 50 µg/mL. The other tested extracts have no significant activity in the methanolic solutions. When extracts were dissolved in 50% methanol (Table 4), only four of them exhibited activity (F, G, H and I), and extract F had the strongest protective effect against CPZ-induced photogenotoxicity. A comparison of all the extracts tested at the same concentration (100 µg/mL) using different solvents is shown in Figure 9.  The results of the umu-test performed with metabolic activation against 2AA-induced genotoxicity are presented in Table 5 and Figure 10. In the highest tested concentration of water solutions (Table 5), only extract A did not exhibit antigenotoxic activity. The highest protective effect was observed for extract F and I, up to 52% of inhibition of IR induced by the 2AA. In the lowest tested concentration of water solutions (100 µg/mL), four extracts exhibited activity (F, G, H, and I), and the most active was extract F. Extract F, when dissolved in methanol (Table 5), was active in all tested concentrations. The methanolic solutions of extracts G and D exhibited a protective effect in the concentration of 100 µg/mL. The increase of 2AA-induced genotoxic effect was observed in the treatment with D in the concentration of 12.5 µg/mL. When extracts were dissolved in 50% methanol (Table 5), only extract I was active in all tested concentrations. Extracts F and G were active in the lowest tested concentration. Extracts B and C exhibited no antigenotoxic potential. Extracts A and D, when dissolved in 50% methanol, exhibited a protective effect in the two tested concentrations. Figure 10 presents a comparison of the activity of different extracts (at the same concentration: 100 µg/mL) dissolved in three different solvents. Extracts F, G, H, and I exhibited higher activity when dissolved in deionized water. Extract D was only active when dissolved in pure methanol or 50% methanol. Each value is expressed as mean ± standard deviation from at least three independent biological evaluations. Bold values denote statistical significance at the p < 0.05 level. A dash "-" denotes that the concentration has not been applied. The extracts resulted from the following treatments were investigated: in the lowest tested concentration. Extracts B and C exhibited no antigenotoxic potential. Extracts A and D, when dissolved in 50% methanol, exhibited a protective effect in the two tested concentrations. Figure 10 presents a comparison of the activity of different extracts (at the same concentration: 100 µg/mL) dissolved in three different solvents. Extracts F, G, H, and I exhibited higher activity when dissolved in deionized water. Extract D was only active when dissolved in pure methanol or 50% methanol.

Discussion
Hairy root cultures, due to their genetic and biosynthetic stability and substantial biomass proliferation capabilities are considered as an abundant system for the production of bioactive compounds [4,22,23]. The optimization of cultural conditions allowed us to achieve the productivity of specific metabolites at levels comparable or even higher than native plants; moreover, hairy roots were reported to biosynthesize phytochemicals that are accumulated only in aerial parts or even absent in the parent plant [24]. This study describes, for the first time, the establishment of Polyscias filicifolia hairy root cultures. Up to now, the hairy root cultures were reported only for several members of the Araliaceae family. In the present study, the development of hairy roots was observed on micropropagated and somatic embryo-derived shoots transformed with A. rhizogenes ATCC 15834 ( Table 1). The application of acetosyringone, which was reported to increase the efficiency of T-DNA transfer into plant cell [2], exerted a beneficial effect on transformation frequency in the current study (Table 1). Jeong et al. [15] reported transformation process of filed-grown Panax ginseng roots by A. rhizogenes KCTC 2744, and Kochan et al. [16] transformed filedgrown seedlings of P. quinquefolium with A. rhizogenes ATCC 15834. In vitro-derived plant material was used for the transformation of P. quinquefolium, where the best response was obtained by the transformation of epicotyls with A. rhizogenes LBA 9402 [18] and Aralia elata with the highest transformation rate observed on root and petiole explants infected with A. rhizogenes ATCC 15834 [20]. In the mentioned reports for the cultivation of hairy roots, mostly modifications of B5 [25] or MS [26] media were used. Whereas, in the present study the highest biomass increase was noted during hairy root cultivation in DCR I [27] ( Figure 4), with an increase to 400 mg/L MgSO 4 and supplemented with L-glutamine 500 mg/L. DCR I medium was originally developed for the cultivation of Taxus × media hairy roots [28].
The previous investigations on hairy root cultures of Araliaceae species focused mostly on the production of ginsenosides. In the current study, the accumulation of several phenolic acids was examined, and among the others CGA demonstrated the highest concentration (1.59 ± 0.06 mg DW) in hairy roots elicited on 1 day with 100 µM MeJA 100 and then transferred to control medium for 30-day cultivation (extract H). However, the CGA content was over 3-fold lower than reported previously in P. filicifolia shoots elicited with 50 µM salicylic acid [10]. Although subsequent HPLC-PDA-ESI-MS analysis showed that CGA is present in the form of its derivative, this is not surprising as CGA was demonstrated to easily transform or degrade to its derivatives [29].
The approach applied in the present study, relying on the elicitation and subsequent transfer of elicited roots for further cultivation to control medium, proved to be superior to elicitation exclusively ( Figure 5). A similar approach was used by Sivanandhan et al. [30] to enhance withanolides production in Withania somnifera hairy root cultures, resulting in a remarkable increase in their production, although the authors did not compare the effectiveness of this approach to the elicitation itself. Nevertheless, this approach allows to obtain a significant increase in hairy root productivity by biosynthesis stimulation without retardation in biomass proliferation, which is often a reported effect of elicitor treatment [31].
Furthermore, in the present study, methanolic extracts resulted from hairy roots cultures were subjected to HPLC-PDA-ESI-MS analysis. The composition of examined extracts varied in relation to the applied treatments. Oleanolic acid was detected only in control cultures up to 2 weeks. While CGA, 5-O-feruoylo-quinic, and 3,5-dicaffeoyl-quinic acids were observed since the third week of culture in control and elicited cultures ( Table 2). Other phenolic acids i.e., caffeic, p-coumaric, ferulic, and sinapic were not detected. Further extract profiling revealed the presence of caffeic and ferulic acid derivatives, which was also reported for methanolic extracts of P. filicifolia shoots [10,14]. These shoot extracts demonstrated considerable antimutagenic, anti-photogenotoxic, and skin cell-protective activity [10,[12][13][14].
In the present study, three types of solvents were used to test whether they would affect the activity due to different degrees of dissolution of the compounds. The influence on cell viability of hairy root methanolic extracts was assessed, and no cytotoxic effect was demonstrated (Figures 6 and 7). This finding corresponds with the previous results reported for P. filicifolia shoot [10] as well callus extracts [12]. Next, photogenotoxicity and genotoxicity of the tested extracts was evaluated by the preliminary umu-test. The results indicate that none of the concentrations tested exhibited a genotoxic effect. Furthermore, in experiments with CPZ, the highest activity of extracts was observed when they were dissolved in water. It could be concluded that observed biological activity was affected by the presence of polar phenolics, which were determined in investigated extracts (Table 4). In addition, almost all extracts, except A, exerted antigenotoxic activity with the highest protective potential determined for extracts F and I, up to 52% of inhibition of IR induced by the 2AA (Figure 9; Table 5). The observed biological activities could be attributed to the presence of caffeic and ferulic acid derivatives, rather than CGA isomer presence; the highest concentration was noted in H extract as caffeic and ferulic acid derivatives, which were reported to be more active [32,33]. It was shown that starting from the 30 day of culture, the enhancement in biosynthetic capacities of hairy roots was observed; in all extracts, 3,5-dicaffeoylquinic and derivative of 5-O-feruoyloquinic acid were determined. Nevertheless, further phytochemical and biological investigations should be carried out to reveal the full application potential of P. filicifolia hairy root extracts.
A voucher specimen of P. filicifolia was deposited at the Department of Pharmaceutical Biology and Medicinal Plants Biotechnology, Faculty of Pharmacy, Medical University of Warsaw, Warsaw, Poland (accession No. FW21/026/1999).

Establishment of Hairy Root Cultures
Genetic transformation was performed with the use of two strains: Agrobacterium rhizogenes: ATCC 15834 and LBA 9402. Bacteria were grown in the dark on solid YMB medium [35] at 26 • C. Prior to transformation, bacteria were incubated for 48 h in liquid medium YMB with or without the addition of 0.2 mg/L acetosyringone (Acs). Acs, dissolved in a drop of 96% ethanol, was added to the autoclaved medium by filtering through a sterile filter (Ø 20 µm pores).
The explants were contaminated by pricking them with a sterile needle dipped in the bacterial suspension. Leaves were infected along the nerve, and petioles at their central part. Plantlets derived from somatic embryos and micropropagated in vitro shoots were pricked several above the second node. After infection, the explants were placed on solid hormone-free MS medium (25 mL of medium in 100 mL Erlenmeyer flasks) or on MS medium supplemented with 0.2 mg/L Acs. After 4 weeks, the percentage of explants on which root growth was observed was calculated, and the number of roots formed per one explant was determined.
After 4 weeks of cultivation on solid MS medium, roots approx. 10 mm long were cut off explants and transferred individually to 100 mL Erlenmeyer flasks containing 25 mL of MS liquid medium supplemented with Biotaxine 500 mg/L. The flasks were placed on the rotary shaker (100 rpm INNOVA 2300 platform shaker, New Brunswick Scientific, Enfield, CT, USA) and cultivated in the dark. After two passages with the antibiotic, the concentration of Biotaxine added to the medium was reduced to 250 mg/L. After four passages, a test was performed for the presence of bacteria in the cultured roots. For this purpose, their fragments were placed on solid YMB and incubated for 48 h at 24 • C in the dark. The lack of bacterial growth indicated the sterility of the obtained transformed roots. The sterile roots were transferred to 250 mL Erlenmeyer flasks containing 50 mL of hormone-free MS liquid medium and routinely subcultured every 4-6 weeks. As a result of the performed transformation, 15 clones of hairy roots were obtained. Finally, only one hairy root line K-1 was selected for further investigation.

DNA Extraction and PCR Analysis
In P. filicifolia roots obtained after transformation of Agrobacterium rhizogenes with ATCC 15834 strain, the presence of rolB, rolC, and virG genes was analyzed using the PCR technique. The primer sequences used to carry out the PCR reaction are shown in Table 6. The PCR reaction was performed using the Phire ® Plant Direct PCR Kit (Finnzymes, Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction. The leaves of non-transformed P. filicifolia donor plant were used as a control. About 3 mm of putative hairy root or control leaves were cut off and suspended in 20 µL of dilution buffer, and 0.5 µL of resulted solution was taken for PCR reaction. The PCR reaction was carried out in a volume of 20 µL, under the conditions according to the protocol. PCR analysis conditions were as follows: one cycle of initial denaturation at 98 • C for 5 min; the next 40 cycles at 98 • C for 5 s, 62 • C for 5 min, and 72 • C for 20 s; and then a final extension at 72 • C for 1 min. The Thermal Cycler Vario (Applied Biosystems, Waltham, MA, USA) for amplification was used. The products were electrophoretically separated on a 1.0% agarose gel (PRONA, Burgos, Spain) with the 1 × TBE buffer using SimplySafe (EURx, Gdańsk, Poland) and UV light visualised (Gel-X Gel Analysis Systems, STI, Poznań, Poland). GeneRuler TM 1 kb Plus DNA ladder (Thermo Scientific, Waltham, MA, USA) molecular weight marker was used to estimate the size of amplification products. Table 6. Sequences of primers used in the PCR reaction [36].

Gene
Forward Primer Reverse Started

Cultivation of Hairy Roots and Elicitation
In order to select the best growth-promoting medium for the cultivation K-1 hairy root for line, the following hormone-free media modifications were tested: (i) DCR (Gupta and Durzan [27] The growth of the root biomass was estimated based on fresh (FW) and dry weight (DW) ( Table S1). The 28-day-old hairy roots of K-1 line were taken out of the flasks and gently pressed on a filter paper, drained, and weighed to record the fresh biomass. Next, the frozen roots were lyophilised (lyophilizer Christ ALPHA1-4 LSC, Osterode am Harz, Germany) until constant weight, and the dry weight (DW) was recorded. The root growth index was calculated based on fresh biomass of roots using the following formula: (final weight-initial weight/initial weight) × 100% [38].

Antigenotoxicity and Anti-Photogenotoxicity Assessment by the umu-Test
The antigenotoxic potential was evaluated against the genotoxic action of 2AA in the presence of metabolic activation. Metabolic activation was performed with an S9 liver fraction of a male Sprague Dawley rat treated with Aroclor 1254 (single dose of 500 mg/kg body weight) in soya oil 5 days before the isolation according to the method described by Maron and Ames [39].
Firstly, the genotoxic potentials of 2AA (10 µg/mL) was measured by the umu-test using Salmonella typhimurium strain TA1535/pSK1002 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DSMZ, Braunschweig, Germany). The umu-test was carried out in the micro-plate variant according to the ISO guideline [40]. β-galactosidase activity of the tested extracts was presented as an induction ratio (IR) calculated as the β-galactosidase activity of the tested extract relative to the negative control. The spectrophotometric measurement was performed at λ = 420 nm to indicate the intensity of the enzymatic reaction, and the optical density at λ = 600 nm indicated the bacteria growth. The measurements were performed with an Asys UVM340 Hightech microplate spectrophotometer. The 1.5-fold and greater increase in the β-galactosidase activity resulted in an IR value of 1.5 or greater and indicated the genotoxicity of the sample. Simultaneously, the genotoxicity of extracts (800 µg/mL or 100 µg/mL) was investigated. Next, the inhibition of 2AA genotoxicity by tested extracts was investigated. Extracts were added to the mutagen at concentrations of 100-800 µg/mL (extracts dissolved in water) or 12.5-100 µg/mL (dissolved in methanol or 50% methanol). Lower concentrations of the solutions were prepared in the case where the solvent was methanol, since methanol is toxic to bacteria.
The rate of antigenotoxicity (%) of the tested sample was calculated as the inhibition of IR induced by 2AA at the particular concentration according to the following Equation: Antigenotoxicity% = (1 − IR genotoxin+sample /IR genotoxin )100% (1) To assess the anti-photogenotoxic effect of tested extracts, the protocol developed by Skrzypczak et al. [41] was applied. Chlorpromazine (CPZ) was used as an agent-inducing genotoxic response under UVA irradiation by forming an unstable promazyl radical, which is able to bind to DNA [42]. CPZ was tested at the concentration of 5 µg/mL by the modified umu-test after UVA irradiation was carried out with the lamp emitting UVA 365 nm (0.231 mW/cm 2 ). The rate of anti-photogenotoxicity was calculated using the same formula as the rate of antigenotoxicity.

Determination of Cytotoxicity Using Neutral Red Uptake Test
The neutral red uptake cytotoxicity test was performed on the basis of modified ISO 10993 guideline Annex A [21] with BALB/c 3T3 clone A31 mammalian cell line (mouse embryonic fibroblasts from American Type Culture Collection). The quantitative estimation of viable cells in tested culture was based on their neutral red uptake in comparison to the results obtained for untreated cells. Dead cells have no ability to accumulate the dye in their lysosomes.
The BALB/c 3T3 cells were seeded in 96-well microplates (10 4 cells/100 µL) in DMEM (Gibco, Thermofisher Scientific, Waltham, MA, USA) culture medium (supplemented with 10% of calf bovine serum, 100 IU/mL penicillin and 0.1 mg/mL streptomycin) and incubated for 24 h (5% CO 2 , 37 • C, >90% humidity). At the end of the incubation, each well was examined under a microscope to ensure that cells form a half-confluent monolayer. After that, culture medium was replaced by the tested solutions, previously sterilized by filtration and 100 times diluted in the fresh culture medium with reduced serum concentration (5%). The highest tested concentration of each sample in the culture medium was 100 µg/mL. Cells were treated with eight dilutions of each solution in a 2-fold dilution series for 24 h (three data points for each variant). Subsequently treatment medium was removed. Cells were washed with PBS and treated with the neutral red medium for 2 h. Then the medium was discarded, and cells were washed with PBS and treated with desorbing fixative (ethanol and acetic acid water solution). The amount of neutral red accumulated by cells was evaluated colorimetrically at 540 nm. As negative and positive controls, fresh culture medium and sodium lauryl sulphate (SLS) were used, respectively. The solvent control was fresh DMEM with 1% of methanol (the concentration of this alcohol in DMEM varies among three variants of tested solutions from 1% to 0%). The percentage of viable cells in each well was calculated by comparing its OD 540 result with the mean result obtained for untreated cells (incubated in the same conditions with fresh culture medium). When the BALB/c 3T3 +4.5 kV for positive mode and −3.5 kV for negative mode; and collision energy in MS2 experiments: 25-35%.
The calibration mixture was used to calibrate the TOF detector of the LCMS-IT-TOF mass spectrometer for high resolution mass spectrometry experiments (HRMS). The sample was prepared by dissolving in the methanol and spinning on a vortex, and the supernatant was transferred to an HPLC injection vial.

Statistical Analysis
The statistical analyses were made on the results of at least three independent biological replicates performed with completely fresh bacteria cultures (all measurements were performed in triplicate or more independent experiments). All data were analyzed using the STATISTICA 13.1 PL software package (StatSoft, Kraków, Poland). For data with normal distribution (tested with Shapiro-Wilk test), Student's t-test was used. Data without normal distribution were analyzed with the nonparametric U Mann-Whitney method. The results were considered to be statistically significantly different at a probability level of p < 0.05.

Conclusions
The effectiveness of root formation on P. filicifolia explants infected with Agrobacterium rhizogenes depended on: the bacterial strain used, the type of explant, and the presence of acetosyringone (Acs) in the medium on which the infected explants were incubated. The approach of elicitation with subsequent hairy root transfer to control medium proved to be the most promoting in relation to the intensification of phenolic acid biosynthesis. Methanolic extracts derived from P. filicifolia hairy roots exhibited no cytotoxic effect while their antigenotoxic and anti-photogenotoxic properties were demonstrated. The most active proved to be dissolved in water extracts derived from hairy roots cultivated in elicited variants of cultures. This could be attributed to the higher level of phenolic compounds such as chlorogenic acid, and dicaffeoylquinic and feruoylcaffeoylquinic acid derivatives, which were more numerous and abundant than in control roots. The results obtained in the current study indicate the future direction of further investigations of P. flilicifolia towards its photoprotective capabilities.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27010186/s1. Figure S1: Confirmation of genetic transformation of Polyscias filicifolia hairy roots; Table S1: Effect of various media modifications on the growth of the K-1 hairy roots of Polyscias filicifolia.